CN104212711A - Electronic sensor and gene detection method based on electronic sensor - Google Patents

Electronic sensor and gene detection method based on electronic sensor Download PDF

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CN104212711A
CN104212711A CN201410138340.0A CN201410138340A CN104212711A CN 104212711 A CN104212711 A CN 104212711A CN 201410138340 A CN201410138340 A CN 201410138340A CN 104212711 A CN104212711 A CN 104212711A
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base
nanoporous
single stranded
stranded dna
measured
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CN104212711B (en
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张世理
吴东平
章贞
克劳斯·安德斯·尤特
拉尔夫·西艾切
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SHANGHAI TURTLE TECHNOLOGY CO., LTD.
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KRAUSS ANDERS UTOR
RALPH XIEQIE
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    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores

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Abstract

The invention relates to the field of biological detection and discloses an electronic sensor and a gene detection method based on the electronic sensor. The electronic sensor comprises ISFET, a nano-wire channel between a source electrode and a drain electrode of the ISFET is provided with a first groove by etching, the bottom of the first groove is provided with nano-pores, or is provided with chemical molecules or is provided with nano-pores and chemical molecules arranged at the nano-pores so that it is avoided that the DNA molecules smoothly slide at a high rate from one side to the other side of the chemical molecules or the nano-pores without control, only after a single-chain DNA to be tested successfully matches a detection basic group, a basic group can be moved forward by the chemical molecules and a matching process of the detection basic group and the basic group of the single-chain DNA to be tested can be accurately detected so that the gene detection result is more accurate, a detection precision is high and detection sensitivity is high.

Description

Electronic sensor and the crto gene method based on electronic sensor
Technical field
The present invention relates to field of biological detection, particularly a kind of electronic sensor and the crto gene method based on electronic sensor.
Background technology
Along with the development of science and technology, thymus nucleic acid (Deoxyribonucleic acid, is abbreviated as DNA) order-checking has become the conventional study project of biological medicine research laboratory, the whole world.Bringing the reason of this phenomenon may be develop rapidly due to sequencing technologies, cause the cost having sequencing equipment to decline, also may be that biochemicals required for checking order and other requisite costs decline.The cost carrying out overall human genome order-checking based on so-called first-generation sequencing technologies meets microelectronic Moore's Law at the rate of descent of calendar year 2001 to 2007 year.As seen from the figure, large-scale parallel sequencing technologies of future generation entered the application stage in 2005, cause cost rate of descent to exceed the several order of magnitude of Moore's Law, but rate of descent had remained basically stable since 2012.The technological revolution of a new round is realize only spending 1000 dollars to complete to check order to whole human genome in the target set at that time.But, in order to make gene order-checking walk out R&D units, and penetrate into medical health department, should well below 1000 dollars to each genomic order-checking into, therefore, it is indispensable for further developing sequencing technologies.
Up to the present, commercially available sequencing technologies is completely all almost fluorescence/chemical labeling the method based on optics.Heavy machine and tediously long operating time, and high cost are the main drawbacks of this technology.Carry out business-like semi-conductor sequencing technologies by ion torrent (Ion Torrent) company in December, 2010 and present revolutionary progress.Semi-conductor sequencing technologies is based on silicon micromachining technology and microelectronic completely, and without the need to mark and opticinstrument.Ion-sensitive field effect transistor (ISFET) schematic diagram (111 is substrate, and 104 is reference electrode) of Ion Torrent system as shown in Figure 1.In figure, magnetic bead 101 carries a large amount of (quantity is greater than 10 4to 10 6) identical single stranded DNA 102, this magnetic bead is placed on and is full of in electrolytical ISFET groove 107.This groove is connected to the fluid pool of the sequentially feeding four kinds of Nucleotide (also referred to as " base ") preset, four kinds of bases are respectively: VITAMIN B4 (A), cytosine(Cyt) (C), guanine (G), thymus pyrimidine (T), not necessarily press the order of A, C, G, T, but in substantially often flowing, each type occurs once.In figure, do not draw fluid pool, but depict the direction 103 that base enters ISFET groove 107.When pairing (namely A-T or C-G is combined, and forms double-strand) appears in the to be measured base of the detection base in fluid on single stranded DNA, a large amount of hydrogen ions will be discharged in a fluid.This will cause soda acid (pH) the value instantaneous variation of ISFET groove Inner electrolysis matter, and the more important thing is the hydrogen ion concentration instantaneous variation of the upper surface of the piled grids causing ISFET.And Surface Hydrogen ionic concn changes the change that will cause ISFET surface potential, thus cause the change of the channel current between ISFET source electrode 105 and drain electrode 106.In order to detect that this changes to greatest extent, can in the surface-coated of piled grids to protonated/deprotonated (or pH value change) especially responsive metal or metal oxide 108.
The great advantage of Ion Torrent technology is that its cost is extremely cheap.Through develop rapidly and the huge investment of decades, the microchip processing of current semiconductor factory all has competitive power in expense and complicacy.On sheet, integrated chip Ion Torrent sensor array and various electronic circuit, calibrates, noise processed, data management etc., makes this emerging technology reduce further holistic cost.
But there is a defect in the chip (up to the present all products) of Ion Torrent company, is exactly in order-checking, usually can produces the error rate higher than optical means.Specifically, the two large major defects that Ion Torrent sequencing technologies exists are:
(1), when single stranded DNA occurring a large amount of repetition base, the precise figures of its repeat length cannot be determined.Such as, GGGGG(repeat length is 5) be 6 with GGGGGG(repeat length), when there is similar repetitions, expection may produce stronger electrical signal, because release more hydrogen ion in single fluid, result in larger pH value and changes.But this change is difficult to determine because long tumor-necrosis factor glycoproteins causes, or a large amount of identical single-stranded DNA copy causes owing to employing.
(2) the reading length (i.e. the total bases of single stranded DNA) of Ion Torrent technology is usually less than 400 base pairs, shorter than other sequence measurements.When to when there is more repetition or have the DNA of many structure variations to check order, longer reading length is favourable.Such as longer order-checking reads length to from the beginning genomic assembling is useful especially.
The sensitivity that these defects are mainly on duty due to planar I SFET phase causes, and its cone had its source on the raceway groove of planar I SFET cuts body micro recessed structure (as shown in Figure 1), because its dominant sidewall areas is not effective detection zone.Therefore, such design should improve, thus obtains better sensitivity.In addition, each magnetic bead employs the copy of a large amount of same DNAs, make the hydrogen ion discharged be difficult in concert with change surface potential, thus cause nonlinear problem, and be difficult to distinguish long repetition.Intuitively, copy with a small amount of DNA, even only use one, checking order, is very favorable, because it not only solves nonlinear problem, it also avoid the mistake that the polymerase chain reaction (PCR) etc. because of DNA cloning causes and surveys.In fact, time-consuming PCR step can be removed.
Semi-conductor sequencing technologies is called as " 2.5 generation ", and individual molecule order-checking is considered to the third generation.Perhaps, most representative is use nanoporous, comprises the nanoporous based on graphene platelet, carries out electronic counting and determine base, thus carry out direct Sequencing, without the need to DNA cloning, and fluorescence/chemical labeling or opticinstrument.The structure of this device and principle of work are very simple, as shown in Figure 2 A and 2B.When single stranded DNA electrophoresis is by nanoporous, when particularly base is by nanoporous, the ion(ic)current between upper and lower electrolytic solution liquid storage tank reduces.By recording the change of these ion(ic)currents, the base number passed through, and by reference to known base spacing, the length of single stranded DNA can be obtained.
But, use nanoporous to be mainly used in up to now measuring DNA length, length can be surveyed and reach several thousand bases, instead of determine the order of sequence, because the difference of the ion blocking-up electric current produced by four kinds of different bases is very little.And ion(ic)current itself is very low, 10 -11to 10 -10ampere (A) order of magnitude, carries out accurate detection to it also very difficult.In addition, the shape of nanoporous and size, DNA base is relative to the position of nanoporous and direction, and ionic nature in electrolytic solution liquid storage tank and ionic strength, and these parameters are also very large on the impact of its result of detection.Between base, the nuance of electronic structure is measured also by no means easy with transverse electric tunnelling current (as shown in Figure 2 B, in figure, 201 is tunnel probe tunneling probe, and 202 is backgate back gate).
Summary of the invention
The object of the present invention is to provide a kind of electronic sensor and the crto gene method based on electronic sensor, make crto gene result more accurate, precision is higher, and sensitivity is higher.
For solving the problems of the technologies described above, the invention provides a kind of electronic sensor, comprising ion-sensitive field effect transistor ISFET, the nanowire channel between the source electrode of described ISFET and drain electrode etches formation first groove;
The bottom of described first groove has one of following structure or combination:
The bottom of described first groove has nanoporous, the bottom of described first groove is placed with chemical molecular;
Wherein, described chemical molecular is used for the movement of controlling gene.
Present invention also offers a kind of crto gene method based on above-mentioned electronic sensor, comprise following steps:
Described first groove is connected By Electrolysis matter liquid storage tank;
Reference electrode is inserted in described By Electrolysis matter liquid storage tank;
Single stranded DNA to be measured is thrown in described By Electrolysis matter liquid storage tank;
Described single stranded DNA to be measured, under the bias voltage effect of described reference electrode, moves to described first bottom portion of groove;
To described By Electrolysis matter liquid storage tank supply detection base; Wherein, supply four kinds of bases with the order preset, supply a kind of base to described By Electrolysis matter liquid storage tank at every turn; Further, before each replacing base, from described By Electrolysis matter liquid storage tank, described first groove, front a kind of base is rinsed away;
After detecting the base pairing success in base and described single stranded DNA, described single stranded DNA to be measured moves a base;
The ion(ic)current detecting described nanoporous is hindered the current peak of appearance or paddy, measures length or the base number of described single stranded DNA to be measured; Wherein, when described base is by described nanoporous, the ion(ic)current of described nanoporous is hindered;
Or in base-pairing events, release hydrogen ions in described nano bowl, detects the curent change between the source electrode of described ISFET and drain electrode.
Compared with prior art, in the present invention ISFET source electrode and drain electrode between nanowire channel on etch formation first groove, the bottom of this first groove or have nanoporous, or placement chemical molecular, or offer nanoporous, and place chemical molecular at nanoporous place, DNA molecular is made to be no longer that the side of chemically molecule or nanoporous unblockedly is at a high speed without sliding into opposite side with controlling, only after single stranded DNA to be measured and detection base pairing success, just move a base forward by chemical molecular, accurately can detect the base-pairing events in detection base and single stranded DNA to be measured, thus make crto gene result more accurate, precision is higher, sensitivity is higher.
In addition, described first groove can be nano bowl, bottom nano bowl, be placed with chemical molecular.
In addition, described first groove can be nano bowl, and the bottom of described nano bowl has nanoporous, and the substrate of described ISFET has the second groove centered by described nanoporous.
In addition, described first groove can be nano bowl, and the bottom of described nano bowl has nanoporous, and the substrate of described ISFET has the second groove centered by described nanoporous, and described nanoporous position is placed with chemical molecular; Wherein, described chemical molecular only allows the double stranded section of gene to move from described nanoporous to the other side.
In addition, the bottom of described nano bowl forms wedge angle knife-edge around described nanoporous, to detect the transverse electric tunnelling current of nanoporous more accurately
In addition, the height of described nano bowl and diameter are less than 200 nanometers; Or the volume of described nano bowl is less than 10 -17rise.Nano bowl has enough little volume can make crto gene result more accurate further, and precision is higher, and sensitivity is higher.
In addition, the diameter of described nanoporous is less than 10 nanometers, with the size match of gene, effectively to detect.
In addition, the thickness of bottom around described nanoporous of described nano bowl is less than 5 nanometers.
In addition, Coating Ions sensitive membrane on the inner side-wall of described nano bowl.Described ion-sensitive mould material can be metal or metal oxide, and these ion-sensitive mould materials are more responsive and adsorption is stronger to hydrogen ion, and adsorption concentration is higher, and sensitivity is good, so just makes measuring result more timely and effective.
In addition, can in conjunction with Organic ionic groups on described ion sensitive membrane.Can activated surface by organic ion, make the electronic sensor of present embodiment more responsive to hydrogen ion.
In addition, described chemical molecular is Phi29DNAP.
In addition, described first groove can be doline; Described funnel-shaped bottom portion has nanoporous; And described first bottom portion of groove forms wedge angle knife-edge around described nanoporous.
In addition, described wedge angle position is placed with chemical molecular; Wherein, described chemical molecular only allows the double stranded section of gene to move from described nanoporous to the other side.
In addition, when detecting the curent change between the source electrode of described ISFET and drain electrode, the base type of supplying when electric current changes is recorded in.
In addition, when detecting that the ion(ic)current of described nanoporous is hindered the current peak of appearance or paddy, be recorded in the base type of supplying when electric current changes.
In addition, after the step detecting the curent change between the source electrode of described ISFET and drain electrode, also following steps are comprised:
According to basepairing rule, the base type of record is found out its complementary base in order, obtain the base sequence of described single stranded DNA to be measured;
Wherein, described basepairing rule is: VITAMIN B4 A and thymus pyrimidine T matches, and guanine G and cytosine(Cyt) C matches.
In addition, after the step of base sequence obtaining described single stranded DNA to be measured, also following steps are comprised:
With reference to known base spacing, according to the base sequence of described single stranded DNA to be measured, obtain the length of described single stranded DNA to be measured.
In addition, in the step detecting the curent change between the source electrode of described ISFET and drain electrode, following sub-step is also comprised:
The number of times that record current changes;
After treating that crto gene is complete, the total degree changed by recorded electric current is as the base number of described single stranded DNA to be measured.
In addition, throwing in the step of single stranded DNA to be measured in described By Electrolysis matter liquid storage tank, only throw in a single stranded DNA to be measured at every turn;
In the step to described By Electrolysis matter liquid storage tank supply base, supply a detection base to described By Electrolysis liquid liquid storage tank at every turn.
Check order by supplying a single single stranded DNA of detection base pair at every turn, its sequencing result is more accurate.Further, owing to only supplying a detection base at every turn, if the curent change between the source electrode of the ISFET detected and drain electrode, so, its result may be only the base pairing success in the detection base and single stranded DNA of supplying; And by the control of Phi29DNAP, even if occur that the base in single stranded DNA repeats, at every turn also can only with double stranded section after a base pairing, therefore, repeat base to long, also can detect exactly.
In addition, throwing in the step of single stranded DNA to be measured in described By Electrolysis matter liquid storage tank, the copy of some identical single stranded DNAs to be measured is thrown in;
Before the step to described By Electrolysis matter liquid storage tank supply base, judge whether the copy of described identical single stranded DNA to be measured all moves to the bottom of described first groove, if not, wait for, until the copy of described identical single stranded DNA to be measured all moves to the bottom of described first groove, then to described By Electrolysis matter liquid storage tank supply base;
In the step to described By Electrolysis matter liquid storage tank supply base, supply some detection bases to described By Electrolysis matter liquid storage tank at every turn; Wherein, the base number of described supply is more than or equal to the copy number of described identical single stranded DNA to be measured.
By multiple detection base and the base pairing in the copy of multiple identical single stranded DNA to be measured, the curent change between the source electrode of ISFET and drain electrode can be made to increase, or the time that the curent change between the source electrode of ISFET and drain electrode continues increases, thus gene sequencing result can be made further more accurate.
Accompanying drawing explanation
Fig. 1 is the ISFET schematic diagram of Ion Torrent system in prior art;
Fig. 2 A is based on the structural representation that the nanoporous direct gene of graphene platelet checks order in prior art;
Fig. 2 B is based on the principle of work schematic diagram that the nanoporous direct gene of graphene platelet checks order in prior art;
Fig. 3 is according to the electronic sensor schematic diagram in first embodiment of the invention;
Fig. 4 is the system schematic of carrying out crto gene according to the electronic sensor in first embodiment of the invention;
Fig. 5 A is according to the electronic sensor diagrammatic cross-section in second embodiment of the invention;
Fig. 5 B is according to the electronic sensor vertical view in second embodiment of the invention;
Fig. 6 is the system schematic of carrying out mrna length and the detection of base number according to the electronic sensor in second embodiment of the invention;
Fig. 7 is the system schematic of carrying out gene sequencing according to the electronic sensor in second embodiment of the invention;
Fig. 8 is a kind of structural representation according to the electronic sensor in third embodiment of the invention;
Fig. 9 is the another kind of structural representation according to the electronic sensor in third embodiment of the invention;
Figure 10 is the system schematic of carrying out gene sequencing according to the electronic sensor in third embodiment of the invention;
Figure 11 is the crto gene system schematic adopted according to the crto gene method in four embodiment of the invention;
Figure 12 is the schema according to the crto gene method in four embodiment of the invention;
Figure 13 is the crto gene system schematic adopted according to the crto gene method in fifth embodiment of the invention;
Figure 14 is the schema according to the crto gene method in fifth embodiment of the invention.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below in conjunction with accompanying drawing, the embodiments of the present invention are explained in detail.But, persons of ordinary skill in the art may appreciate that in each embodiment of the present invention, proposing many ins and outs to make reader understand the application better.But, even without these ins and outs with based on the many variations of following embodiment and amendment, each claim of the application technical scheme required for protection also can be realized.
First embodiment of the present invention relates to a kind of electronic sensor, as shown in Figure 3, this electronic sensor comprises ion-sensitive field effect transistor (ISFET), nanowire channel 304 between its source electrode 302 and drain electrode 303 etches formation first groove, and the bottom of this first groove has one of following structure or combination: the bottom of the first groove has nanoporous, the bottom of the first groove is placed with chemical molecular; Wherein, chemical molecular is used for the movement of controlling gene.That is, the bottom of this first groove or have nanoporous, or placement chemical molecular, or offer nanoporous, and place chemical molecular at nanoporous place, DNA molecular is made to be no longer that the side of chemically molecule or nanoporous unblockedly is at a high speed without sliding into opposite side with controlling, only after single stranded DNA to be measured and detection base pairing success, just move a base forward by chemical molecular, accurately can detect the base-pairing events in detection base and single stranded DNA to be measured, thus making crto gene result more accurate, precision is higher, and sensitivity is higher.
In the present embodiment, the first groove can be nano bowl, and the substrate 301 that the bottom of this nano bowl has nanoporous 306, ISFET has groove centered by nanoporous 306.
The volume of nano bowl does enough little, relatively can increase the concentration of ion in ionogen.Such as, the height of nano bowl and diameter are less than 200 nanometers, or the volume of nano bowl is less than 10 -17rise.Specifically, the normal blood pH value of people is 7.4, corresponds to quite low proton concentration, about every cubic centimetre 24 × 10 12, namely can only find 1 proton in the nanocubes of the 360nm length of side.Be less than 100 nanometers with the height of nano bowl and diameter, or its volume is less than 10 -18rise (attolitre, namely 10 6cubic nanometer) be example, although only have a proton in the nano bowl of little volume like this, but the concentration of its correspondence almost increases by 50 times than in proton mean dispersion situation in the normal blood of people, correspondingly, in volume little like this, produce a proton will correspondingly make pH value be changed to 5.7 from 1.7, or make potential shift 110 millivolts (mV), considerably beyond the resolution limit (0.1-1mV) of typical ISFET.Therefore, nano bowl has enough little volume can make crto gene result more accurate further, and precision is higher, and sensitivity is higher.
The electronic sensor of present embodiment can be described as " 3NANO sensor " (being called for short 3NANO), comprises three nano level concepts: nano wire, nano bowl and nanoporous.Custom-designed SOI substrate has SiO 2/si 3n 4/ SiO 2storehouse as insulation layer (as in Fig. 3 301), for follow-up making nanoporous is ready.The thickness of surface silicon layer can be 200 nanometers, first makes and uses the nano wire ISFET based on current ISFET preparation technology, and new beamwriter lithography (EBL) technology also can be adopted to make nanostructure.The nano-wire array of wide 50 to 300 nanometers can be used for forming optimum nano bowl, and the thickness of nano bowl (i.e. the height of nano bowl) can obtain from the thickness 200nm adjustment of surface silicon layer easily.Under the doping type of nano wire and concentration also adjust to optimum polarity and bias condition.By suitably arranging the gate bias voltage of electrolytic solution, the hydrogen ion discharged in base-pairing events can be made as far as possible to move to sensitive surface (i.e. ion sensitive membrane 305), instead of move to the direction away from nano bowl.
When seeing from the top down, nano bowl profile can be combination that is circular, oval or several different basic configuration, can be selected the accurate shape of sensitivity the best, workability optimum by emulation.The internal surface of nano bowl needs to be designed to minute surface, to eliminate corrosion damage.Thermal oxidation technology can be adopted to form the SiO with most stiff stability 2/ silicon interface, its interfacial configuration and thermostability are all better, highly sensitive, and acoustic noise control capability is stronger.The top one deck that ion sensitive membrane will be deposited, refers to hereafter.In addition, the 3NANO sensor of present embodiment has the source/drain of optimization, insulation layer, and passivation and metallic contact district, to reduce external disturbance as far as possible.
The nanoporous be positioned at bottom nano bowl can by the silicon nitride film of a few nanometer thickness, adopt beamwriter lithography (EBL) mode top-down approach to be formed nanoporous that diameter reaches 20 nanometers, also can adopt hard mask and conformal thin-film deposition (such as ald ALD) to combine to be formed the nanoporous that diameter is less than 10 nanometers.The pore size of nanoporous can be reduced further by ALD, but the effective film thickness around nanoporous is also need to control, and that is, the bottom of nano bowl is less than 5 nanometers at the thickness at nanoporous edge.Therefore, can the number of times of control ALD as required, make the film thickness around the aperture of nanoporous and nanoporous all adjust to suitable value.
Can Coating Ions sensitive membrane on the inner side-wall of nano bowl.The nano bowl be etched in nano-wire transistor channel can inject ionogen, and such design makes the inner side of almost whole nano bowl, except nanoporous, comprises upright side walls, is all sensitive surface.Certainly, also can only Coating Ions sensitive membrane in upright side walls, the same sensing effect can be reached.When the electronic sensor of application present embodiment carries out crto gene, due to Coating Ions sensitive membrane on the inner side-wall of nano bowl, so the inner side-wall of whole nano bowl all can be used for detecting hydrogen ion concentration, base-pairing events in detection base and single stranded DNA to be measured is accurately detected, thus make crto gene result more accurate, precision is higher, and sensitivity is higher.
The material of the ion sensitive membrane in the present embodiment can be metal or metal oxide, and metal more common at present has gold, platinum, palladium etc., and more common metal oxide has hafnium oxide (HfO 2), titanium dioxide (TiO 2), aluminum oxide (Al 2o 3) or tantalum pentoxide (Ta 2o 5) etc., these ion-sensitive mould materials are more responsive and adsorption is stronger to hydrogen ion, and adsorption concentration is higher, and sensitivity is good, so just makes measuring result more timely and effective.This ion sensitive membrane can make film by the mode of thermooxidizing, chemical vapour deposition or ald above-mentioned materials, and these modes are all relatively simple comparatively speaking, requires also not high, can reduce the preparation cost of whole device to film-forming apparatus.
In addition, it is worth mentioning that, can also in conjunction with Organic ionic groups on ion sensitive membrane, such as hydroxide radical OH-ion, hydrosulphuric acid hydrogen root SH-ion.These organic ions can activated surface, makes the electronic sensor of present embodiment more responsive to hydrogen ion.
In addition, what deserves to be explained is, in the present embodiment, semiconducter substrate can be P type or N-type, if semiconducter substrate is P type, then what formed by doping is exactly source electrode and the drain electrode of N-type; If semiconducter substrate is N-type, then what formed by doping is exactly source electrode and the drain electrode of P type.In figure 3, be P-type semiconductor substrate, form the source electrode of N-type and drain electrode by doping, and define P type heavy doping (in figure P+ region) at source electrode and drain electrode.Because the mobility of electronics is much larger than the mobility in hole, therefore do source electrode and drain electrode with the N-type doping that electronics is majority carrier, much better than by current capacity; In addition, say from control angle, N-type ISFET can open with positive voltage, uses more convenient.
Adopt the electronic sensor of present embodiment to carry out the system of crto gene, as shown in Figure 4, nanoporous position is placed with chemical molecular 401 to its structure, and nano bowl connects By Electrolysis matter liquid storage tank 402, second groove and connects lower ionogen liquid storage tank 403.
There are some technology at present, by measuring stream of electrons by being integrated with the nano-wire fet of nanoporous, having adopted and improving reliability with the basecount be associated that is shifted.In the art, it is crucial that the speed how to be shifted by nanoporous control DNA.Utilize light or called after " archaeal dna polymerase of Phi29 " (referred to as Phi29DNAP) can be proved to be feasible by speed control.Phi29DNAP is also used in other order-checking, the most famous is in zero mould optical waveguides (ZMW) technology, successfully use the enzyme of this mystery, control to move at the single stranded DNA of single base level, each successful base pairing causes single stranded DNA base to move.That is, Phi29DNAP or similar chemical molecular etc. is added in nanoporous ingress, the existence of Phi29DNAP makes DNA molecular be no longer unblockedly from the side of nanoporous (figure above nanoporous, By Electrolysis matter liquid storage tank 402 side) slide into opposite side (in figure below nanoporous, lower ionogen liquid storage tank 403 side) without control at a high speed.Under reasonable terms, Phi29DNAP only allows the double stranded section of DNA pass through, and that is, only after the detection base A, G, C, T base pairing success of single stranded DNA to be measured and inflow, DNA just moves a base by Phi29DNAP toward below.
Apply above-mentioned principle, when carrying out gene sequencing, upwards throw in single stranded DNA in ionogen liquid storage tank, supply detection base (indicating with pentagram) from fluid pool upwards ionogen liquid storage tank, direction, as shown in figure 404, detects the base pairing in base A, C, G, T and single stranded DNA, after successful matching, single stranded DNA originally becomes double-strand, and under the control of Phi29DNAP, downward ionogen liquid storage tank moves a base.In each base-pairing events in single stranded DNA, produce hydrogen ion 406, be discharged in nano bowl, make electrolytical pH value generation temporal variation in nano bowl.Under the bias voltage effect of reference electrode 405, hydrogen ion moves in (in figure direction shown in 407) to the inwall of nano bowl, make the channel current between the source electrode of ISFET and drain electrode also temporal variation occur thereupon, by detecting the channel current change between the source electrode of ISFET and drain electrode, the base pairing successful moment can be obtained exactly.In brief, when being hindered by the ion(ic)current of nanoporous with survey, the change (occurring current peak or paddy) of (when just in time certain base is by nanoporous) measures length or the base number of DNA; Or, by the change of ISFET electronic current and the synchronous processing flowing into detection base sequential, determine the order of DNA; Also by determining the order of DNA to the synchronous processing of the change of nanoporous ion(ic)current and inflow detection base sequential; Rear both also can be combined, and can improve the accuracy rate (cross-check) of order-checking.
In addition, meriting attention is that Yeast Nucleic Acid (RNA) forms long chain molecule by ribonucleotide through the condensation of phosphide key.A ribonucleic acid molecule is by phosphoric acid, and ribose and base are formed.The base of RNA mainly contains 4 kinds, i.e. VITAMIN B4 (A), guanine (G), cytosine(Cyt) (C), uridylic (U), and wherein, U instead of the T in DNA.With above set forth detect similar about single stranded DNA, the electronic sensor of present embodiment also can be used for detecting RNA, difference is that detection base type is A, G, C, U, the chemical molecular similar with Phi29DNAP is placed in nanoporous ingress, can control RNA from nanoporous side to the movement of opposite side, length, base number, the principle of subsequent detection and DNA carry out to RNA similar, do not repeat one by one at this.
Second embodiment of the invention relates to a kind of electronic sensor, second embodiment is roughly the same with the first embodiment, main difference part is: in the first embodiment, first groove is nano bowl, in this second embodiment, first groove is doline, funnel-shaped bottom portion has nanoporous, and wedge angle (knife-edge) is formed around nanoporous on the bottom of the first groove, its structure as shown in Figure 5 A and 5B, wherein, Fig. 5 A is the diagrammatic cross-section of the ISFET of present embodiment, and Fig. 5 B is the vertical view of the ISFET of present embodiment.
Specifically, the ISFET of present embodiment is by the Si in Fig. 2 A 3n 4/ SiO 2nanoporous has replaced to the wedge angle nanoporous ISFET that is only had a few nanometer thickness, to detect the transverse electric tunnelling current of nanoporous more accurately.Adopt system shown in Figure 6, when can be hindered by the ion(ic)current of nanoporous with survey, the change (occurring current peak or paddy) of (when just in time certain base is by nanoporous) measures length or the base number of DNA.Adopt system shown in Figure 7, be placed with chemical molecular in wedge angle position; This chemical molecular only allows the double stranded section of gene mobile to the other side (shown in figure downside) from nanoporous one side (shown in figure upside), by the synchronous processing to the change of ISFET electronic current and inflow detection base sequential, determines the order of DNA; Also by determining the order of DNA to the synchronous processing of the change of nanoporous ion(ic)current and inflow detection base sequential; Rear both also can be combined, and can improve the accuracy rate (cross-check) of order-checking.Its ultimate principle and the first embodiment similar, this is no longer going to repeat them.
Second embodiment of the invention relates to a kind of electronic sensor, and the second embodiment is roughly the same with the first embodiment, and main difference part is: in the first embodiment, and the first groove is nano bowl, has nanoporous bottom nano bowl; In this second embodiment, the first groove is nano bowl, but does not open nanoporous bottom nano bowl, directly places chemical molecular, and its structure as shown in Figure 8.Specifically, the bottom of nano bowl is stacked structure, has stacked gradually insulation layer 801, Si medium layer 802 and ion sensitive membrane 803; Chemical molecular 804 is placed on ion sensitive membrane.
Or the first groove is nano bowl, opens nanoporous bottom nano bowl, be sitting in position at nanoporous and place chemical molecular, its structure as shown in Figure 9.In figure, 301 is substrate, and 304 is the nano-channel between the source electrode 302 of ISFET and drain electrode 303, and 401 is chemical molecular.In the present embodiment, can in nano bowl whole inwall Coating Ions sensitive membrane, also can only in upright side walls Coating Ions sensitive membrane 305.
The electronic sensor of present embodiment is adopted to carry out the system of crto gene as shown in Figure 10, under reference electrode 405 acts on, the single stranded DNA to be measured rendered in By Electrolysis matter liquid storage tank moves bottom nano bowl, upwards inject detection base (representing with pentagram) in ionogen liquid storage tank, when detecting the base pairing success in base and single stranded DNA to be measured, release hydrogen ions in nano bowl, makes electrolytical pH value generation temporal variation in nano bowl.Under the bias voltage effect of reference electrode, hydrogen ion moves to the inwall of nano bowl, make the channel current between the source electrode of ISFET and drain electrode also temporal variation occur thereupon, by detecting the channel current change between the source electrode of ISFET and drain electrode, the base pairing successful moment can be obtained exactly.Its ultimate principle and the first embodiment similar, this is no longer going to repeat them.
Third embodiment of the invention relates to a kind of crto gene method, is the crto gene method of the electronic sensor based on the first embodiment, specifically carries out single single stranded DNA detection, and as shown in figure 11, detection flow process as shown in figure 12 for its concrete detection principle.
Specifically, first nano bowl is connected By Electrolysis matter liquid storage tank, groove connects lower ionogen liquid storage tank; Between By Electrolysis matter liquid storage tank and the ionogen of lower ionogen liquid storage tank, be biased voltage, upwards throw in single stranded DNA to be measured in ionogen liquid storage tank, only throw in a single stranded DNA to be measured at every turn; This single stranded DNA to be measured is under the effect of bias voltage, and by nanoporous, downward ionogen liquid storage tank quick travel, at nanoporous place, is stopped by chemical molecular.
Upwards ionogen liquid storage tank supply detection base, each supply one detection base; Wherein, supply four kinds of bases with the order preset, upwards ionogen liquid storage tank supplies a kind of base at every turn, and, before each replacing base, separate in matter liquid storage tank, nano bowl, groove, lower ionogen liquid storage tank from power on and front a kind of base is rinsed away.
After detecting the base pairing success in base and single stranded DNA, the downward ionogen liquid storage tank of single stranded DNA to be measured moves a base;
The ion(ic)current detecting nanoporous is hindered the current peak of appearance or paddy, measures length or the base number of single stranded DNA to be measured; Wherein, when base is by nanoporous, the ion(ic)current of nanoporous is hindered.
In base-pairing events, to release hydrogen ions in nano bowl, therefore, can detect the curent change between the source electrode of ISFET and drain electrode, and be recorded in the base type of supplying when electric current changes.Or, when detecting that the ion(ic)current of nanoporous is hindered the current peak of appearance or paddy, be recorded in the base type of supplying when electric current changes.Then, according to basepairing rule, the base type of record is found out its complementary base in order, obtains the base sequence of single stranded DNA to be measured; Wherein, basepairing rule is: VITAMIN B4 A and thymus pyrimidine T matches, and guanine G and cytosine(Cyt) C matches.
In addition, after the base sequence obtaining single stranded DNA to be measured, with reference to known base spacing, according to the base sequence of single stranded DNA to be measured, the length of single stranded DNA to be measured can be obtained.Further, in the process detecting the curent change between the source electrode of ISFET and drain electrode, can also the number of times that changes of record current; After treating that crto gene is complete, the total degree changed by recorded electric current is as the base number of single stranded DNA to be measured.
Present embodiment is checked order by the single single stranded DNA of each supply one detection base pair, and its sequencing result is more accurate.Further, owing to only supplying a detection base at every turn, if the curent change between the source electrode of the ISFET detected and drain electrode, so, its result may be only the base pairing success in the detection base and single stranded DNA of supplying; And by the control of Phi29DNAP, even if occur that the base in single stranded DNA repeats, at every turn also can only with double stranded section after a base pairing, therefore, repeat base to long, also can detect exactly.
Four embodiment of the invention relates to a kind of crto gene method, is the crto gene method of the electronic sensor based on the first embodiment, specifically carries out multiple single stranded DNA detection, and as shown in figure 13, detection flow process as shown in figure 14 for its concrete detection principle.
Specifically, first nano bowl is connected By Electrolysis matter liquid storage tank, groove connects lower ionogen liquid storage tank, voltage is biased between By Electrolysis matter liquid storage tank and the ionogen of lower ionogen liquid storage tank, upwards throw in single stranded DNA to be measured in ionogen liquid storage tank, throw in the copy of some identical single stranded DNAs to be measured; This single stranded DNA to be measured is under the effect of bias voltage, and by nanoporous, downward ionogen liquid storage tank quick travel, at nanoporous place, is stopped by chemical molecular.
Before upwards ionogen liquid storage tank supplies base, judge whether the copy of identical single stranded DNA to be measured all moves to nanoporous, if not, wait for, until the copy of identical single stranded DNA to be measured all moves to nanoporous, more upwards ionogen liquid storage tank supplies base.
Upwards during ionogen liquid storage tank supply detection base, the some detection bases of each supply; Wherein, supply four kinds of bases with the order preset, upwards ionogen liquid storage tank supplies a kind of base at every turn, and the base number of supply is more than or equal to the copy number of identical single stranded DNA to be measured.Further, before each replacing base, separate in matter liquid storage tank, nano bowl, groove, lower ionogen liquid storage tank from power on and front a kind of base is rinsed away.
After detecting the base pairing success in base and single stranded DNA, the downward ionogen liquid storage tank of single stranded DNA to be measured moves a base; The ion(ic)current detecting nanoporous is hindered the current peak of appearance or paddy, measures length or the base number of single stranded DNA to be measured; Wherein, when base is by nanoporous, the ion(ic)current of nanoporous is hindered.
In addition, similar with the 3rd embodiment, also can carry out the detections such as order-checking to single stranded DNA to be measured, its principle of work is similar, for avoiding repetition, does not repeat them here.
In the present embodiment, due to multiple detection base and the base pairing in the copy of multiple identical single stranded DNA to be measured, therefore, there are two kinds of situations:
(1) multiple detection base simultaneously with the base pairing in single stranded DNA, if now successful matching, its hydrogen ion produced is by more than the hydrogen ion produced during single single stranded DNA, so ISFET source electrode and drain electrode between curent change also can increase, thus be convenient to this curent change be detected, gene sequencing result can be made further more accurate.
(2) base pairing when multiple detection base is different and in single stranded DNA, if now successful matching, then can be within a certain period of time, continue to produce hydrogen ion, thus the time making the curent change between the source electrode of ISFET and drain electrode continue is also longer, also be conducive to this curent change being detected, gene sequencing result can be made further more accurate.
The step of various method divides above, just in order to be described clearly, can merge into a step or splitting some step, being decomposed into multiple step, when realizing as long as comprise identical logical relation, all in the protection domain of this patent; To adding inessential amendment in algorithm or in flow process or introducing inessential design, but the core design not changing its algorithm and flow process is all in the protection domain of this patent.
Persons of ordinary skill in the art may appreciate that the respective embodiments described above realize specific embodiments of the invention, and in actual applications, various change can be done to it in the form and details, and without departing from the spirit and scope of the present invention.

Claims (24)

1. an electronic sensor, comprises ion-sensitive field effect transistor ISFET, it is characterized in that, the nanowire channel between the source electrode of described ISFET and drain electrode etches formation first groove;
The bottom of described first groove has one of following structure or combination:
The bottom of described first groove has nanoporous, the bottom of described first groove is placed with chemical molecular;
Wherein, described chemical molecular is used for the movement of controlling gene.
2. electronic sensor according to claim 1, is characterized in that, described first groove is nano bowl.
3. electronic sensor according to claim 2, is characterized in that, is placed with described chemical molecular bottom described nano bowl.
4. electronic sensor according to claim 2, is characterized in that, the bottom of described nano bowl has nanoporous, and the substrate of described ISFET has groove centered by described nanoporous.
5. electronic sensor according to claim 4, is characterized in that, described nanoporous position is placed with chemical molecular; Wherein, described chemical molecular only allows the double stranded section of gene to move from described nanoporous to the other side.
6. electronic sensor according to claim 2, is characterized in that, the height of described nano bowl and diameter are less than 200 nanometers;
Or the volume of described nano bowl is less than 10 -17rise.
7. electronic sensor according to claim 4, is characterized in that, the diameter of described nanoporous is less than 10 nanometers.
8. electronic sensor according to claim 4, is characterized in that, the bottom of described nano bowl is less than 5 nanometers at the thickness at described nanoporous edge.
9. electronic sensor according to claim 2, is characterized in that, Coating Ions sensitive membrane on the inner side-wall of described nano bowl.
10. electronic sensor according to claim 9, is characterized in that, the material of described ion sensitive membrane is metal or metal oxide.
11. electronic sensors according to claim 10, is characterized in that, in conjunction with Organic ionic groups on described ion sensitive membrane.
12. electronic sensors according to claim 10, is characterized in that, the bottom of described nano bowl is stacked structure, have stacked gradually insulation layer, Si medium layer and ion sensitive membrane;
Described chemical molecular is placed on described ion sensitive membrane.
13. electronic sensors according to claim 1, is characterized in that, described chemical molecular is Phi29DNAP.
14. electronic sensors according to claim 1, is characterized in that, described first groove is doline; Described funnel-shaped bottom portion has nanoporous;
Described first bottom portion of groove forms wedge angle knife-edge around described nanoporous.
15. electronic sensors according to claim 14, is characterized in that, described wedge angle position is placed with chemical molecular; Wherein, described chemical molecular only allows the double stranded section of gene to move from described nanoporous to the other side.
16. electronic sensors according to claim 1, is characterized in that, the material of described nanoporous be following one of arbitrarily:
Silicon Si, silicon nitride SiNx, silicon oxide sio 2, diamond, Graphene graphene.
17. 1 kinds, based on the crto gene method of the electronic sensor as described in any one of claim 1 to 16, is characterized in that, comprise following steps:
Described first groove is connected By Electrolysis matter liquid storage tank;
Reference electrode is inserted in described By Electrolysis matter liquid storage tank;
Single stranded DNA to be measured is thrown in described By Electrolysis matter liquid storage tank;
Described single stranded DNA to be measured, under the bias voltage effect of described reference electrode, moves to the bottom of described first groove;
To described By Electrolysis matter liquid storage tank supply detection base; Wherein, supply four kinds of bases with the order preset, supply a kind of base to described By Electrolysis matter liquid storage tank at every turn; Further, before each replacing base, from described By Electrolysis matter liquid storage tank, described first groove, front a kind of base is rinsed away;
After detecting the base pairing success in base and described single stranded DNA, described single stranded DNA to be measured moves a base;
The ion(ic)current detecting described nanoporous is hindered the current peak of appearance or paddy, measures length or the base number of described single stranded DNA to be measured; Wherein, when described base is by described nanoporous, the ion(ic)current of described nanoporous is hindered;
Or in base-pairing events, release hydrogen ions in described nano bowl, detects the curent change between the source electrode of described ISFET and drain electrode.
18. crto gene methods according to claim 17, is characterized in that, when detecting the curent change between the source electrode of described ISFET and drain electrode, are recorded in the base type of supplying when electric current changes.
19. crto gene methods according to claim 17, is characterized in that, when detecting that the ion(ic)current of described nanoporous is hindered the current peak of appearance or paddy, are recorded in the base type of supplying when electric current changes.
20. crto gene methods according to claim 18 or 19, is characterized in that, after the step detecting the curent change between the source electrode of described ISFET and drain electrode, also comprise following steps:
According to basepairing rule, the base type of record is found out its complementary base in order, obtain the base sequence of described single stranded DNA to be measured;
Wherein, described basepairing rule is: VITAMIN B4 A and thymus pyrimidine T matches, and guanine G and cytosine(Cyt) C matches.
21. crto gene methods according to claim 20, is characterized in that, after the step of base sequence obtaining described single stranded DNA to be measured, also comprise following steps:
With reference to known base spacing, according to the base sequence of described single stranded DNA to be measured, obtain the length of described single stranded DNA to be measured.
22. crto gene methods according to claim 17, is characterized in that, in the step detecting the curent change between the source electrode of described ISFET and drain electrode, also comprise following sub-step:
The number of times that record current changes;
After treating that crto gene is complete, the total degree changed by recorded electric current is as the base number of described single stranded DNA to be measured.
23. crto gene methods according to claim 17, is characterized in that, throwing in the step of single stranded DNA to be measured in described By Electrolysis matter liquid storage tank, only throw in a single stranded DNA to be measured at every turn;
In the step to described By Electrolysis matter liquid storage tank supply base, supply a detection base to described By Electrolysis liquid liquid storage tank at every turn.
24. crto gene methods according to claim 17, is characterized in that, throwing in the step of single stranded DNA to be measured in described By Electrolysis matter liquid storage tank, throw in the copy of some identical single stranded DNAs to be measured;
Before the step to described By Electrolysis matter liquid storage tank supply base, judge whether the copy of described identical single stranded DNA to be measured all moves to the bottom of described first groove, if not, wait for, until the copy of described identical single stranded DNA to be measured all moves to the bottom of described first groove, then to described By Electrolysis matter liquid storage tank supply base;
In the step to described By Electrolysis matter liquid storage tank supply base, supply some detection bases to described By Electrolysis matter liquid storage tank at every turn; Wherein, the base number of described supply is more than or equal to the copy number of described identical single stranded DNA to be measured.
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